In type II diabetes, the unregulated production of glucose from the liver, due to hepatic insulin resistance, is a major cause of deleterious chronic hyperglycemia. One of the key determinants of hepatic glucose production is the transcriptional activity of the PEPCK gene, which encodes the rate limiting enzyme in the gluconeogenic pathway. The expression of the PEPCK gene is known to be regulated by various metabolic signals including glucagon and insulin. It has also been demonstrated by us and others to be regulated by the cellular energy-sensing kinase AMPK. This important metabolic kinase is traditionally believed to be the key sensor for energy homeostasis in the cytoplasm. We have developed a hypothesis, which forms the basis of the proposal, whereby AMPK also carries out an important function in the nucleus, regulating the expression of genes involved in metabolic homeostasis. Specifically, we propose that AMPK functions as transcriptional cofactor, directly regulating PEPCK gene expression. This hypothesis is supported by preliminary data demonstrating that the catalytic isoform of the kinase is present in the nucleus and, more importantly, is bound to the chromatin at the promoter of the endogenous PEPCK gene. The goals of the proposed research are: 1) to characterize the interaction of AMPK with the PEPCK gene in vivo using the chromatin-IP procedure, 2) to identify the set of nuclear proteins that interact with AMPK using a candidate factor approach as well as tandem-affinity-purification method to identify unknown interacting proteins, and 3) to test one model for how AMPK might regulate PEPCK gene transcription. In this model the kinase acts by phosphorylating the transcription factor HNF4-alpha (a known AMPK target and PEPCK transcriptional regulator) and alters its interaction with the coactivator PGC1-alpha. Results generated from these studies will contribute to our understanding of the role of AMPK in regulating gene transcription in response to metabolic signals. [unreadable] [unreadable] [unreadable]